Diagnostic and Prognostic Value of SHOX2 and SEPT9 DNA Methylation and Cytology in Benign, Paramalignant and Malignant Pleural Effusions

Abstract

Pleural effusions (PE) are a common clinical problem. The discrimination between benign (BPE), malignant (MPE) and paramalignant (PPE) pleural effusions is highly important to ensure appropriate patient treatment. Today, cytology is the gold standard for diagnosing malignant pleural effusions. However, its sensitivity is limited due to the sometimes low abundance of tumor cells and the challenging assessment of cell morphology in cytological samples. This study aimed to develop and validate a diagnostic test, which allows for the highly specific detection of malignant cells in pleural effusions based on the DNA methylation biomarkers SHOX2 and SEPT9. A quantitative real-time PCR assay was developed which enabled the accurate and sensitive detection of SHOX2 and SEPT9 in PEs. Cytological and DNA methylation analyses were conducted in a case control study comprised of PEs from 114 patients (58 cases, 56 controls). Cytological analysis as well as SHOX2 and SEPT9 methylation resulted in 100% specificity. 21% of the cases were cytologically positive and 26% were SHOX2 or SEPT9 methylation positive. The combined analysis of cytology and DNA methylation resulted in an increase of 71% positively classified PEs from cancer patients as compared to cytological analysis alone. The absolute sensitivity of cytology and DNA methylation was not determinable due to the lack of an appropriate gold standard diagnostic for distinguishing between MPEs and PPEs. Therefore, it was unclear which PEs from cancer patients were malignant (containing tumor cells) and which PEs were paramalignant and resulted from benign conditions in cancer patients, respectively. Furthermore, DNA methylation analysis in PEs allowed the prognosis of the overall survival in cancer patients (Kaplan-Meier analysis, log rank test, p = 0.02 (SHOX2), p = 0.02 (SEPT9)). The developed test may be used as a diagnostic and prognostic adjunct to existing clinical and cytopathological investigations in patients with PEs of unclear etiology.

Current status and future perspectives of circulating cell-free DNA methylation in clinical diagnostics

“…This semiquantitative result is then transformed into a qualitative result (testpositive and test-negative). However, an advantage of DNA methylation testing is the simplicity and high accuracy, robustness and repeatability of a quantitative analysis using a calibrator and an adapted ΔΔCT method [49,50]. Reporting the quantitative test result therefore might help to improve the test performance in the future.…”

Diagnosis of Lung Cancer by SHOX2 Gene Methylation Assay

“…This product has been used clinically in combination with the Applied Biosystems 7500 Fast or Fast DX Real-Time polymerase chain reaction (PCR) System. Although the assay has been performed clinically with bronchoalveolar lavage fluid samples collected at bronchoscopy [13][14][15], its applications with other types of samples are also under investigation, and these include lung cancer tissue [17], lymph node tissue around lung cancer [19], pleural effusion (hydrothorax) [20,21], and plasma [16]. These studies showed that histology or cytology samples obtained for pathological or cytological examinations can also be used for the SHOX2 gene methylation assay to confirm suspected diagnosis.…”

“…However, a large discrepancy has been observed in sensitivity between samples. The sensitivity for lung tissue/lymph nodes samples, bronchial lavage samples and plasma samples was above 94 % [17,19], between 64-78 % [13][14][15] and about 60 % [16], respectively, and the positivity rate with pleural effusion samples was less than 40 % [20,21]. Therefore, it shows a clear trend in sensitivity: lung cancer tissue & lymph node tissue [ bronchoalveolar lavage fluid [ plasma.…”

“…The SHOX2 assay with pleural effusion samples is distinct from that with other samples and has special application in tumor staging and surgery. The purpose of the assay with pleural effusion samples is to distinguish pleural effusions caused by cancer (malignant pleural effusions) and pleural effusions caused by benign conditions, such as congestive heart failure and other benign lung diseases, as it was not designed for discriminating between patients with and without cancer [20]. This purpose is very different from that with other types of samples.…”

“…Several clinical trials have found it to be a sensitive biomarker for lung cancer detection with bronchoalveolar lavage fluid samples [13][14][15]. Studies have also proved it to be very effective for confirming diagnosis with various types of samples, no matter if it was used alone or in combination with histological or cytological examinations [16][17][18][19][20][21]. Although the commercialized assay is currently only permitted to be used for auxiliary diagnosis with bronchoalveolar lavage fluid samples from bronchoscopy, its future application with other samples is promising.…”